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Current Opinion in Colloid & Interface Science 16 (2011) 228–237
Contents lists available at ScienceDirect
Current Opinion in Colloid & Interface Science
j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c o c i s
Fate of polymeric nanocarriers for oral drug delivery
Laurence Plapied, Nicolas Duhem, Anne des Rieux, Véronique Préat ⁎
Université catholique de Louvain, Louvain Drug Research Institute, Unité de pharmacie galénique, Avenue Mounier, 73 UCL 7320, 1200 Brussels, Belgium
a r t i c l e
i n f o
Article history:
Received 27 October 2010
Received in revised form 22 December 2010
Accepted 23 December 2010
Available online 8 January 2011
Keywords:
Oral drug delivery
Polymeric nanoparticles
Polymeric micelles
a b s t r a c t
This review will focus on two polymeric nanocarriers: nanoparticles and micelles that have been studied for
oral drug delivery at preclinical level. Their potential for oral drug delivery will first be illustrated. Then their
mechanisms of uptake and their fate after oral delivery will be discussed. Future directions for oral delivery
with nanocarriers will be analyzed with a special emphasis on optimal properties. The recent advances
highlight the need to tune and to control their design with a good balance in their physicochemical properties
and suggest that more sophisticated nanosystems will be developed for the oral delivery of drugs,
biopharmaceuticals and vaccines, thanks to (i) the development of biocompatible polymers with tailored
properties for oral drug delivery and formulation of nanocarriers, (ii) the understanding of cellular uptake
mechanisms of polymeric nanocarriers, (iii) the novel techniques to study the fate of nanocarriers, polymers
and drugs in the body and (iv) the identification of new ligands for targeted oral delivery.
Major recent advances: Recent advances in the (i) development of biocompatible polymers with tailored
properties for oral drug delivery and nanocarrier formulation, (ii) the understanding of cellular uptake
mechanisms of polymeric nanocarriers (iii) the new techniques to study fate of nanocarriers, polymers and
drugs in the body and (iv) the identification of new ligands for targeted oral delivery have promoted the
development of novel polymeric carriers for the oral delivery of drugs, biopharmaceuticals and vaccines.
© 2011 Elsevier Ltd. All rights reserved.
1. Introduction
1.1. Oral administration: advantages and drawbacks
Oral administration is the preferred route for drug delivery. It is
patient-friendly, painless and easy for self-medication. Compared to
parenteral delivery, it suppresses risk of disease transmission, reduces
cost and increases patient compliance. It allows flexible and controlled
dosing schedule. It is particularly convenient for chronic therapy [1–4].
The intestinal epithelium is specialized in nutriment absorption but
provides a physical barrier to drug absorption. It is composed of absorptive
enterocytes for a large part sprinkled by mucus-producing Goblet cells,
endocrine and Paneth cells. Immunocompetent cells (B and T lymphocytes, dendritic cells) are located in the lamina propria beneath the
epithelium except for intraepithelial lymphocytes and dendritic cells that
are inserted between the enterocytes. The “Follicle Associated Epithelium”
(FAE) forms the interface between the luminal environment and the
lymphoid tissue associated to the gut (GALT) composing Peyer's patches
that are mostly found in the ileum [5]. The FAE contains enterocytes and M
cells that are structurally dissimilar to enterocytes. They possess less
mucous glycocalyx, less microvilli and differences in the dominance and
⁎ Corresponding author. Tel.: +32 2 764 73 09; fax: +32 2 764 73 98.
E-mail addresses: [email protected] (L. Plapied),
[email protected] (N. Duhem), [email protected] (A. des Rieux),
[email protected] (V. Préat).
1359-0294/$ – see front matter © 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.cocis.2010.12.005
the pattern of the cell surface receptors [6]. M cells play a role of
gatekeepers that continuously take up and internalize material from the
lumen and transport it into the underlying lymphoid tissue [7].
Oral bioavailability of drugs is strongly influenced by their properties.
Solubility and permeability are two important parameters for their
absorption via passive diffusion. The Biopharmaceutic Classification
System defines four categories of drugs based on their solubility and
their permeability [8]. A drug that is administered orally must survive
transit through the chemical and enzymatic gastrointestinal (GI) liquids,
cross the mucus layer and the epithelium before being absorbed. If most
small molecules are resistant to the harsh environment of the GI tract and
can be absorbed, the intestinal barrier limits the oral absorption of
macromolecules such as proteins, vaccines or nucleic acids. Hence,
protective vehicles to avoid destruction in the GI tract and potentially
enhance oral absorption are needed.
The unique characteristics of the GI tract can be exploited for
optimizing formulations aiming at enhancing drug absorption, e.g.
changes in pH and microflora for targeting the colon or mucoadhesion
for increasing residence time of a drug.
1.2. Nanotechnology
The European Science Foundation defines nanomedicines as nanometer size scale complex systems, consisting of at least two components,
one of which being the active ingredient. Although mainstream
nanotechnology explores particles between 1 and 200 nm in diameter,
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L. Plapied et al. / Current Opinion in Colloid & Interface Science 16 (2011) 228–237
the size of individual particles tested for drug delivery of therapeutic and
imaging agents may range from 2 to 1000 nm [9,10].
Nanotechnology brings some advantages to the drug delivery field
in general and oral drug delivery in particular. It allows (i) the delivery
of poorly water-soluble drugs, (ii) the targeting of drugs to specific
part of the gastrointestinal tract, (iii) the transcytosis of drugs across
the tight intestinal barrier and (iv) the intracellular and transcellular
delivery of large macromolecules [11]. Nanomedicines can increase
efficacy, specificity, tolerability and therapeutic index of corresponding
drugs [12]. They must be stable, non-toxic, non-thrombogenic, nonimmunogenic, non-inflammatory, biodegradable, avoid uptake by
reticulo-endothelial system and should be applicable to various
molecules such as small drugs, proteins, vaccines or nucleic acids [10–
14]. Among the nanomedicines, nanocarriers such as polymeric
nanoparticles or micelles have provided a promising approach to obtain
desirable biopharmaceutical and pharmacokinetic properties for
medicines.
1.3. Scope of the review
This review will focus on two polymeric nanocarriers: nanoparticles
and micelles. Their potential for oral drug delivery will first be illustrated.
Then their mechanisms of uptake and their fate after oral delivery will be
discussed. Future directions for oral delivery with nanocarriers will be
analyzed with a special emphasis on optimal properties.
The characteristics of drug-loaded polymeric nanocarriers, their
potential therapeutic applications, their putative mechanisms of
absorption and their fate after oral drug delivery are summarized in
Table 1.
2. Polymeric nanoparticles and micelles
2.1. Polymeric nanoparticles
Nanoparticles include nanocapsules and nanospheres. Nanocapsules
are vesicular systems in which a drug is confined to a cavity surrounded
by a polymer membrane, whereas nanospheres are matrix systems in
which the drug is physically and uniformly dispersed [10].
Different methods can be selected to prepare nanoparticles
depending on the nature of the polymer as well as on the drug to be
encapsulated [15]. Most of the methods involve the use of organic
solvents, heat, sonication or vigorous agitation which may be harmful to
biopharmaceuticals. Nanoparticles formation can be also based on
electrostatic interactions. These polyelectrolyte complexes do not
require aggressive conditions during preparation, therefore minimizing
possible damage to drug during formation [14].
A large panel of biodegradable polymers is available to form
nanoparticles. They can be either natural or synthetic [16]. Natural
229
materials used for oral delivered nanoparticles include chitosan,
dextran, gelatine, alginate, agar among which chitosan is the most
popular [17,18]. It is a widely available modified natural carbohydrate
polymer prepared by the partial N-deacetylation of chitin. Chitosan is
biocompatible, non-toxic and mucoadhesive. The properties of
chitosan are greatly influenced by its molecular weight and degree
of deacetylation. The presence of reactive functional groups in
chitosan provides great opportunity for chemical modification,
which allows a wide range of derivatives possessing unique
properties. Chitosan has limited solubility at pH above 6.5. Derivatives
of chitosan, synthesized by introducing alkyl groups to amine groups,
for instance quaternized derivatives of chitosan, are permanently
positively charged and overcome solubility drawbacks of chitosan.
Chitosan is able to increase intestinal permeability by opening tight
junctions. Chitosan can form polyelectrolyte complexes of approximately 200 to 400 nm [14,18–20]. Overall, it is evident that chitosan
and its derivatives are useful carriers [21]. Currently, dietary
supplements of chitosan are tested in clinical trials to lower blood
cholesterol but no clinical trials with chitosan nanoparticles are
ongoing [22]. To a lesser extent, dextran, gelatine and alginate are also
used for medical applications due to their biocompatibility.
The main synthetic polymers used for oral drug delivery are: poly
(lactide) (PLA), poly(glycolide) (PGA), poly(lactide-co-glycolide) (PLGA),
poly(cyanoacrylates) (PCA), polyethylenimine (PEI) or polycaprolactone
(PCL) [14,17]. PLA, PLGA and PCL are biocompatible and biodegradable by
hydrolysis in the body in monomeric units. PCL degradation is slower than
PLGA making it more appropriate for long-term delivery [14]. PCA are
degraded by esterases in biological fluids.
In order to increase nanoparticle interactions with the intestinal
mucosa, surface can be modified by adsorption or grafting of
hydrophilic molecules that confer hydrophilicity (e.g. PEG) or
bioadhesivity (e.g. chitosan). Moreover, delivery of medicines to
target specific cells, diseases or areas of the intestine can be achieved
by grafting of ligands such as antibodies, glycoproteins or peptides to
the surface of nanoparticles [13]. Micro-organisms-derived adhesive
factors (flagellin, invasins), vitamins [23,24], and carbohydrates [25]
are also used as targeting ligands (Fig. 1). Optimization of ligand
density on the nanoparticles surface must allow tissue penetration
and cellular uptake resulting in optimal therapeutic efficacy [11].
2.2. Polymeric nanoparticles in oral drug delivery
The use of various polymeric materials and their processing
enables the modulation of nanoparticle physicochemical properties
(e.g. hydrophobicity or surface charges), the extent of drug loading,
the drug release profile as well as their biological behaviour. The large
versatility of these systems allows the oral delivery of a wide variety
of drugs [26]. Compared to other colloidal nanocarriers such as
Table 1
Overview of polymeric nanocarrier for oral active delivery [3,13].
Physicochemical characteristics
Active (drug, protein, vaccine)
Polymeric micelles
Nanoparticles
▪
▪
▪
▪
▪
▪
▪ ~ 200 nm
▪ Static structure
▪ Targeting ligand on the surface
mainly grafted on PEG
▪ Encapsulated
▪ Preferably encapsulated rather than adsorbed
▪ Hydrophilic or hydrophobic
▪ Hydrophilic or hydrophobic
▪ Low or large MW
▪ Low or large MW
▪ Carrier
▪ Carrier
▪ Targeting to specific cells
▪ Protection of fragile drugs
—Mainly enterocytes
▪ Solubilisation of poorly soluble drugs
—M cells (vaccine)
▪ Mainly endocytosis
▪ Receptor mediated endocytosis,
mainly clathrin-dependent
▪ Depending on the polymer and nanoparticle stabilities, transcytosis of nanoparticles or of drug
~ 20–50 nm
Dynamic structure
Hydrophilic corona
Hydrophobic core
In the hydrophobic core
Mainly poorly soluble drugs
Therapeutic applications
▪ Solubilisation of poorly soluble drugs
Mechanism(s) of absorption
▪ Passive diffusion of the drug
▪ Endocytosis of drug-loaded micelles
▪ Polymer partly absorbed both
as unimers and micelles
Fate of the carrier
▪ ~ 200 nm
▪ Static structure
▪ Variable surface properties
Targeted nanoparticles
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Fig. 1. Nanoparticle surface modifications to enhance their uptake by enterocytes and M cells. Non-specific strategies: mucoadhesive polymers coating or forming nanoparticles; PEG
chains forming a hydrophilic protective layer stabilizing the colloidal formulation (passive targeting). Specific strategies: ligands coupled or not coupled to a PEG chain specific for
receptors on enterocytes, M cells or both.
liposomes, lipid-based systems or most micelles, most nanoparticles
are more stable in the GI tract. The main mechanisms involved in the
enhanced drug absorption by polymeric nanoparticles are: (i)
protection of the drug from the harsh environment of the GI tract,
(ii) prolongation of the residence time in the gut by mucoadhesion,
(iii) endocytosis of the particles and/or (iv) permeabilizing effect of
the polymer [13]. Moreover, specific delivery can be achieved by
targeted nanoparticles. In contrast to microparticles that are too large
to pass through the epithelium and must release their payload in the
GI tract, nanoparticles can be taken up and cross the intestinal barrier
[2,13,27]. Several physicochemical parameters seem to influence
translocation of particles across the epithelium, including surface
hydrophobicity, polymer nature and particle size [13].
Encapsulation in polymeric nanoparticles has been reported for
poorly soluble drugs though simpler and cheaper systems exist. It has
also extensively been studied for the oral delivery of peptides and
proteins [13,18]. In particular, many efforts have been made to develop a
successful oral insulin delivery using polymeric nanoparticles [28–30].
Polymeric nanoparticles, e.g. amine-modified graft polyesters, chitosan
nanoparticles [31] or thiolated trimethylchitosan nanoparticles [32],
have been shown to increase bioavailability of insulin (a decrease in
blood glucose level up to 70% of the initial value). These nanoparticles
protect insulin against degradation and facilitate the uptake of insulin
either associated or not associated to the nanoparticles [29]. Mucoadhesion whereby a prolonged retention in intestinal tract translates to
cumulative insulin release and absorption, seems important [30]. The
pharmacological activity of insulin-loaded nanoparticles has been
established almost exclusively in preclinical models. The low number
of clinical trials on oral delivery of insulin by polymeric nanocarriers
results from the poor oral bioavailability of insulin and from the lack of
control of the dose of insulin which is absorbed by the patient. Hence,
the feasibility, both in terms of controlled hypoglycaemic activity in
patients and of marketing and production cost of nanoparticulate oral
delivery of insulin remains to be investigated.
The specificities of M cells have also been exploited to orally
deliver bioactive molecules, specifically vaccines as these cells are
specialized for antigens sampling in mucosal immunity. Nanoparticles
mimicking pathogen structure could be used for oral drug delivery,
especially for oral immunisation. Pattern recognition receptors (PRRs)
on the surface of M cells that have been identified as important in
antigen transcytosis include toll-like receptor-4, and α5β1 integrin
[6]. A few specific ligand of human M cells have been identified in vitro
or in vivo [7,13,33]. Therefore, grafting ligand that specifically target M
cells specific receptors, including PRRs, is particularly attractive for
oral vaccine delivery. However, variation in M cells populations and
receptors is an issue that needs to be addressed. Indeed M cell
populations vary with respect to species, anatomical location,
developmental stage and as a consequence of exogenous factors. In
humans, the number of M cells along the GI tract increases at puberty
and declines thereafter. There is growing interest to discover if M cells
in different species, including human, might have a common set of
conserved apical membrane target protein. Some distinct epitopes
have been described for individual species but there is still no broadly
applicable conserved species-independent label [7].
Another potential application is the oral delivery of polymeric
nanoparticles in the scope of inflammatory bowel disease. Researchers have designed polymeric nanoparticles targeting inflamed tissue
by exploiting its specificities like an elevated level of mucus
production, an enhanced permeability and the presence of an
increased number of immune-related cells [34]. Untargeted nanoparticles, pH sensitive nanoparticles or targeted nanoparticles grafted
with a ligand identified by phage display [35] have been shown to
locally enhance the delivery of anti-inflammatory drugs and to
improve the evolution of experimental colitis [34].
2.3. Polymeric micelles
Surfactants and amphiphilic polymers can self assemble above the
critical micellar concentration (CMC) in colloidal dispersions of
molecular aggregates of approximately 20 to 100 nm called micelles.
The hydrophilic moiety, usually PEG, forms the corona of the micelles
whereas the hydrophobic moiety forms their core [3]. In contrast to
nanoparticles which display a static and stable structure, micelles
form a dynamic structure: surfactant or amphiphilic copolymers
forming the micelles can be exchanged with free surfactants or
unimers (Table 1). The amphiphilic copolymers provide better kinetic
and thermodynamic stability than conventional surfactants. The
hydrophobic core of micelles can solubilise poorly soluble drugs and
partly protect the drug from the aqueous environment. Therefore, the
use of polymeric micelles for oral drug delivery has been mainly
restricted to the delivery of poorly soluble drugs. Depending on the
aqueous solubility of the polymers, polymeric micelles can be formed
either by simple direct dissolution in water or by dissolving drug and
polymer in organic solvents before solvent elimination by dialysis or
evaporation [1,3].
For oral drug delivery, the copolymers used to form micelles
should (i) spontaneously self-assemble in water, (ii) enhance drug
solubility by several orders of magnitude and provide high loading
efficiency, (iii) remain stable upon dilution in the GI tract, (iv) be
biocompatible and non toxic and (v) easy to synthesize at large scale.
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Low molecular weight polyester-PEG, such as PLA-PEG or PCL-PEG
have been extensively studied for drug micellisation [3] but their use
as oral drug delivery systems remains limited due to the necessity of
organic solvents and sophisticated manufacture.
Pluronic block copolymers also known under non-proprietary
name “poloxamer” consists of hydrophilic poly(ethylene oxide) (PEO)
and hydrophobic poly(propylene oxide) (PPO) blocks arranged in a
A–B–A triblock structure and characterized by different hydrophilic–
lipophilic balances [36]. Pluronics solubilise drugs and enhance drug
transport across the intestinal barriers [37].
MonomethylPEG750-poly(caprolactone-co-trimethylcarbonate)
(PEG-p(CL-co-TMC)) has been developed to improve the oral
bioavailability of poorly water-soluble drugs. This polymer spontaneously self-assembles in micelles in aqueous solutions, increases the
solubility of poorly soluble drugs by 1 to 3 orders of magnitude [38]
and enhances their oral bioavailability [39,40].
α-Tocopherol is a lipophilic vitamin whose biocompatibility and
potential to deliver both poorly soluble and water-soluble drugs make
tocols attractive as drug delivery vehicles [41]. Pegylation of vitamin E
forming derivatives such as tocopherol polyethylene glycol succinate
(TPGS) provides water-solubility and surfactant properties to the
vitamin E and allows the formation of micelles [42].
pH-sensitive polymeric micelles have been investigated to
minimize the initial burst release in the acidic stomach and release
the drug in a molecularly dispersed form when the inner core of the
polymer ionizes at pH N5. These pH-sensitive polymers contain in
their hydrophobic block a pH-sensitive unit such as acrylic acid (AA)
moieties and hydrophobic non ionizable units for self-assembly.
Alternatively, the external corona can be ionized. Pluronic-PAA
copolymers self-assemble into micelles with hydrophobic cores of
dehydrated PPO and multilayered coronas of hydrophilic PEO and
partially ionized PAA segments. The ionizable carboxyls in the
micellar corona facilitate mucoadhesion that enhances the residence
time of the micelles [1].
3. Fate of nanoparticles and micelles
Much work has been carried out on the design and preparation of
polymeric nanocarriers (nanoparticles and micelles) [1–4,10–15,27–
30,37,38]. Their use as oral delivery system has been rather extensively
studied but the behaviour of these nanocarriers in the GI tract has been
given less attention. Hence, the mechanisms of nanocarrier absorption
will be discussed and examples of the fate of nanocarriers will be given.
The intestinal mucosa is a major barrier to overcome for oral drug
delivery [43]. Once the barrier of mucus is crossed, drug-loaded
nanoparticles have to be transported across the intestinal epithelium
via the paracellular pathway, transcytosis and/or receptor-mediated
transcytosis by enterocytes or M cells. Whether drugs are absorbed
and delivered in the systemic circulation free or encapsulated will
strongly influence their pharmacokinetics and their biodistribution.
3.1. Stability in the GI tract
After oral administration, the nanocarriers will encounter the
physico-chemical environment of the GI tract. These biological fluids
will influence the stability of particles even before they enter in
contact with the intestinal cells. Polymeric nanocarriers can be
degraded due to the variation of pH levels and the presence of
enzymes or bile salts. Hence, in vitro tests in gastric and intestinal
simulated fluids are of primary importance to investigate if, how and
where the active molecules will be released.
The composition of the nanocarrier will strongly influence its
stability in the GI tract. If nanoparticles are prepared with insoluble
polymers, they will neither be immediately degraded nor rapidly
release the drug. In contrast, water soluble polymers which form
polyelectrolyte nanoparticles will be influenced by the pH or ionic
231
strength and are more likely to be destabilized. Even if their kinetic
stability is better than surfactant micelles, polymeric micelles
concentration should remain above the CMC upon dilution in the GI
tract to avoid release in the GI tract and should be exposed to an ionic
strength below their flocculation point [10].
3.2. Mucoadhesion and the barrier of mucus
The nanocarriers must adhere to the mucus and must cross the
mucus layer. Drugs delivered to mucosal surfaces are usually efficiently
removed by mucus clearance mechanisms [44]. The luminal surface of
mucosal tissues is protected by a highly viscoelastic layer [45]. However,
protective mucus coatings typically trap and rapidly remove foreign
particles from the GI tract [46]. Viruses can diffuse through mucus and
penetrate to the epithelium even though they have to diffuse
“upstream” through mucus that is being continuously secreted. This
ability is mainly due to a smaller size than the mucus mesh spacing and a
non-mucoadhesive surface. A strategy to overcome the mucus barrier
would be to develop nanoparticles mimicking these viruses [44,45]. So,
nanoparticles must be small (b200 nm) to diffuse through the mucus
and avoid elimination by mucilliary clearance [47].
Many groups focussed their researches on mucoadhesive nanoparticles. Indeed, strong interactions with mucus could increase retention
at mucosal surface. These interactions are driven by hydrogen bonding,
Van der Waals interactions, polymer chain interpenetration, hydrophobic forces and electrostatic/ionic interactions [46]. Nanoparticle surface
charges seem to play an important role in particle uptake. Indeed
negatively charged intestinal mucosa, due to the glycocalix, attracts
positively charged nanoparticles. Cationic polymers like chitosan and its
derivatives, coating of nanoparticles with cationic groups or with groups
binding to mucin (like thiol) form particles with an increased residence
time in the GI tract.
Besides mucoadhesion to increase residence time, diffusion in the
mucus is critical. The transport of drug vectors across the barrier of
mucus has significant implications for the development of novel drug
delivery systems; however it is in general poorly characterized.
Quantitative and qualitative information such as diffusivity, viscoelasticity, pore size, velocity, directionality and transport mode can be
determined from particle trajectories. There is a potential for
development of oral drug delivery systems utilizing fast-diffusing
nanoparticle carriers with engineered surface coatings. PEG coating of
particles surface is a way, among others, to ensure rapid nanoparticle
transport in mucus. PEG was first used to increase stability of particles
but it makes also particle more hydrophilic and hence modulates their
bioadhesive properties [46,48]. Dense coating with PEG effectively
minimizes adhesive interactions between nanoparticles and mucins,
allowing penetration of nanoparticles [44,49]. The surface chemistry
of the particle will then influence its transport through the mucus. The
particle mobility also seems to be strongly dependent on surface
charges. Crater and Carrier [50] demonstrated a 20–30 times faster
diffusion for anionic particles in comparison with cationic ones.
Transport rates were inversely related to particle surface potentials,
with negatively charged particles displaying significantly higher
transport rates than near neutral, or positively charged particles
whose transport was severely limited, likely by particle aggregation
and electrostatic adhesive interactions with mucin fibres [50].
In conclusion, a balance between mucoadhesion and mucus
penetration is important for an efficient oral delivery. Since particles
immobilized by mucus are cleared from the mucosal tissue, the
elaboration of mucus-penetrating systems is a priority to improve
mucosal drug delivery. Nanoparticles must be small enough to avoid
significant steric inhibition by the fibre mesh and should avoid
adhesion to mucin fibres [46]. Concomitantly, they should be
mucoadhesive to prolong retention time and contact with intestinal
mucosa.
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3.3. Mechanisms of cellular uptake of nanocarriers
Understanding the fate of nanoparticles in cells constituting the
intestinal epithelium is critical for development of new efficient oral
nanocarriers. To study the mechanisms of nanoparticle transport
across the intestinal barrier, in vitro models have been used. The Caco2 monolayer which can be used to assess nanocarrier permeability is
particularly helpful as (i) only diffusion, endocytosis/transcytosis of
the drug and/or the carrier is evaluated (ii) specific modulators or
inhibitors of uptake mechanisms can be employed (iii) colocalization
of the nanosystems with specific endocytosis markers can be
achieved. Moreover, coculture of Caco-2 cells with HT29 cells to
produce mucus [51] or with Raji cells to mimic the FAE [33] has been
developed. Ussing chambers have also been used [52]. Combination of
both quantitative analysis to measure nanoparticle transport and
confocal microscopy to localize nanoparticles gives a better overview
of the process. Very recently, fluorescent nanoparticles have been
visualized in patients with ulcerative rectocolitis by endoscopy. A
preferential localization in the ulcerated rectum has been reported
[53]. The use of quantum dots as cellular tracking probe of
nanocarriers has also improved the visualization of particles transport
[54].
Particle absorption could involve both paracellular and transcellular
routes (Table 2). However, paracellular route is limited because it utilizes
less than 1% of the mucosal surface area. Furthermore, junctional
complexes (tight or adherens junction proteins) restrict or completely
block the passage between cells of macromolecules or aggregates larger
than approximately 1 nm. Therefore, it is generally admitted that
polymeric nanocarriers do not diffuse through the intestinal barrier by
paracellular route. New potential modulators of the junctional proteins
are developed to reversibly open membranous barriers and improve drug
delivery by the paracellular way [43]. They can act directly or indirectly on
tight junction components. Some act on proteins either by interacting
with extracellular domains of the tight junctions proteins or with a surface
receptor, activating a cascade leading to disassembly of tight junctions.
Some will modulate tight junctions by chelating calcium inducing
disruption of adherens junctions and tight junctions via activation of
protein kinase C. However, exact mechanisms of some modulators are not
known [43]. Interestingly, several nanocarrier components have been
reported to open tight junctions and increase paracellular transport of
drugs. Though controversial, chitosan and its derivatives, in solution
seems more active to open tight junctions than formulated as
nanoparticles [18].
Two main nanoparticle endocytosis mechanisms have been described:
phagocytosis which is restricted to M cells and phagocytic immune cells
and pinocytosis (Fig. 2). The endocytic pathways differ with the size of the
endocytic vesicle, the nature of the cargo and the mechanisms of vesicle
formation [55]. The heterogeneity in endocytic pathways ensures that
different cargoes are internalized to specific intracellular locations and
processes [56]. Internalization of particles by pinocytosis can occur by
different mechanisms: macropinocytosis, clathrin-mediated endocytosis
(CME), caveolae-mediated endocytosis (CvME) and clathrin- and
caveolae-independent endocytosis [16,55]. Macropinocytosis is a transient process while micropinocytosis (clathrin-dependent, caveolaemediated, clathrin- and caveolae-independent endocytosis) is a constitutive pathway. Clathrin-coated vesicles and macropinosomes fuse with
endolysosomes whereas caveolae-coated vesicles can escape endolysosomes and lead to direct exocytosis [57]. Several endocytic mechanisms
often take place simultaneously [16]. The description of all endocytosis
and phagocytosis mechanisms has been recently reviewed (Fig. 2)
[16,55,58,59]. Macropinocytosis involves Rho-family GTPases triggering
the actin-driven formation of membrane protrusions. These protrusions
collapse onto and fuse with the plasma membrane to form macropinosomes of a size above 1 μm. The intracellular fate of macropinosomes
varies depending on cell type. In most case, it will be acidified and shrink
or may fuse with lysosomal compartment or recycle their content to the
surface [55]. Clathrin is a three-legged structure, called a triskelion, formed
by three clathrin heavy chains, each with a tightly associated clathrin light
chains [60]. CME occurs either via specific receptor–ligand interaction or
via non-specific endocytosis [16]. Specific CME involves the concentration
of high-affinity transmembrane receptors and their bound ligands into
“coated pits” on the plasma membrane, the main assembly unit being
clathrin, a cytosolic coat protein. Coated pits invaginate until fission of
vesicle requiring the GTPase dynamin to form endocytic vesicles (100 to
120 nm) that are encapsulated by a polygonal clathrin coat and carry
concentrated receptor–ligand complexes into the cell. When CME
Table 2
Oral targeting of nanoparticles (illustrative, non exhaustive list).
Cells targeted
Active targeting ligand
Target
Enterocytes/mucus
Mannose
Mannose binds lectins expressed on
lymphoid and non-lymphoid cells
(predominantly on antigen presenting
cells APC)
Glycoproteins and glycolipids of
enterocyte membranes
N-acetyl-D-glucosamine and sialic
acid on both M cells and intestinal
absorptive cells
Lectins
— Wheat Germ Agglutinin (WGA)
Vitamins
— Thiamine
— B12
Enterocyte receptors
Flagellin
M cells
Galectine 9 ligand
Mucoadhesion
Toll-like receptors 5 agonist on APCs
Lectin: galectine 9
Injured intestinal epithelium
UEA-1 ligand
Integrin ligands (RGD, LDV…)
Peptide sequence T18 (LTHPQDSPPASA)
Lectin: UEA-1
Integrins
Injured intestinal epithelium
Observations
References
[11,83–85]
Good resistance to
acidic pH and enzymatic
degradation
Binding decreases from
jejunum to ileum
(diminution of mucin)
Good intestinal absorption
Anti-phagocytic activity
pH dependent, Na+ independent,
carrier mediated mechanisms
Bioadhesive in distal gut
complex with intrinsic factor (IF)
if recognized by IF specific receptor
[54]
[86–89]
[23,90]
[24,91]
[92,93]
Up-regulation of galectin-9 on FAE
Highly expressed in
immune tissues
Specific targeting of M cells
By phage screening
[94]
[94]
[70,95]
[35]
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L. Plapied et al. / Current Opinion in Colloid & Interface Science 16 (2011) 228–237
233
Fig. 2. Pathways of particle endocytosis in cells. The endocytic pathways differ with the size of the endocytic vesicle, the nature of the cargo and the mechanisms of vesicle formation
[55].
involves non-specific charge or hydrophobic interactions with cell
membrane, it is a non-specific adsorptive pinocytosis. In both cases,
after formation of the vesicles, early endosomes are acidified by ATPdependent proton pumps and fusionned with late endosomes where the
cargo will be degraded [16,55,58]. Caveolin is a dimeric protein that binds
cholesterol, inserts as a loop into the inner leaflet of the plasma
membrane, and self associates to form a striated caveolin coat on the
surface of the membrane invaginations [55]. In most cells, caveolae are
only slowly internalized (half time N20 min). So the formation of the small
vesicles contributes to bulk fluid phase uptake. CvME consists in formation
of flask-shaped invaginations of the plasma membrane in cholesterol and
sphingolipid-rich microdomains [55]. These invaginations are static
structures with a size of 50 to 100 nm [55,58,61,62] at the plasma
membrane [63,64]. In opposition with CME, CvME is a highly regulated
process involving complex signalling pathways. The fission of caveolae
from membrane, mediated by GTPase dynamin generates cytosolic
caveolar vesicles which do not contain any enzymatic cocktail. Therefore,
this pathway is employed by many pathogens to escape degradation by
lysosomal enzymes [16]. Ligands known to be internalized by CvME
include folic acid, albumin and cholesterol [58]. Caveolae is one type of
cholesterol-rich microdomain but other “rafts” exist. They are small
structures, approximately 40 to 50 nm in diameter, that diffuse freely on
the cell surface. These microdomains allow endocytosis independent of
clathrin- and caveolin-coated pits. These small rafts can be captured by
and internalized within any endocytic vesicle. The mechanisms that
govern caveolae- and clathrin-independent endocytosis remain poorly
understood. Nonetheless, it is likely that each of these pathways fulfils
unique functions in the cell and varies mechanistically not only in how the
vesicles are formed, but also in terms of which cargo molecules they
transport, to what intracellular destination their cargo is delivered and
how their entry is regulated [55].
The pathway(s) used for internalization of nanoparticles will
depend on physicochemical characteristics of the particle and the cell
type [16]. It is now accepted than the most commonly used
nanocarriers, chitosan and PLGA particles utilize clathrin-dependent
endocytosis [59]. Human M-like cells in vitro model has been well
described [33] and allows quantitative and mechanistic transport
studies of particles. By this way, it has been demonstrated that the
presence of M cell enhances particle transport (Fig. 3A). It is well
established that uptake by enterocytes and M cells is size-dependent
[33]. Small particles (b50–100 nm) can be translocated by endocytosis through enterocytes. Larger particles are more likely taken up
and translocated by M cells.
The pathways used for polymeric micelles and unimers also
depend on the polymers and their aggregation state. Pluronic 85
unimers enter epithelial cells through caveolae-dependent and
independent-pathways whereas micelles are internalized exclusively
through CME [59]. PEG-p(CL-co-TMC) unimers can diffuse passively
through model lipid bilayers (PAMPA) [65] whereas micelles are
taken up by endocytosis [66].
As mentioned above, endocytosis can be mediated by ligand
binding to receptors. It could be interesting to enhance nanocarrier
transport by specifically targeting some of these receptors. Indeed,
grafting or coating nanocarriers with ligands binding specific
receptors can enhance their internalization and transport. Endocytosis
of targeted nanoparticles occurs mostly by CME [16,57,67]. Table 2 gives
a non exhaustive list of receptors that have been targeted to increase
nanocarrier transport by intestinal cells or M cells. These receptors are
Fig. 3. Transport of nanoparticles across Caco-2 cell mono-cultures and FAE model
(co-cultures of Caco-2 cells and Raji cells). A. Transport of chitosan (CS/TPP/OVA),
trimethylchitosan (TMC/TPP/OVA), PLGA (PLGA/OVA) nanoparticles and ovalbumin
(OVA) [81]. B. Transport of PEGylated PLGA-based nanoparticles (PLGA NP) and RGD
targeted nanoparticles (PLGA-RGD NP) in the presence or absence of an inhibitor of
β1 integrin [96].
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L. Plapied et al. / Current Opinion in Colloid & Interface Science 16 (2011) 228–237
either well known receptors expressed by intestinal cells or new targets
that have been identified by phage display for specific and non specific
translocation [68,69].
Among them, lectins are proteins that bind sugar reversibly and
are involved in many cell recognition and adhesion processes. Some
lectins interact with adhesion molecules on enterocytes and M cell
membranes. Their conjugation to polymeric nanoparticles significantly increases the transport across the intestinal mucosa mostly by
clathrin-mediated uptake [14,67]. PEGylatyed PLGA nanoparticles
grafted with RGD or RGD peptidomimetic targeting α5β1 integrin
overexpressed at M cells apical surface enhanced transport, as
compared to untargeted nanoparticles [70] (Fig. 3B). The grafting of
new M cell homing peptides identified by phage display on
nanoparticles is associated with increased delivery to M cells and
enhanced transport [68,71]. Thiamine-coated nanoparticles show a
strong capacity to be captured by Peyer's patches [23]. Grafting
vitamin B12 allows internalization through the “intrinsic factor”
specific receptor by a clathrin-mediated uptake [24]. Access Pharmaceuticals recently reported that its novel Cobalamin-coated insulin
containing nanoparticle formulations delivered orally provided a
pharmacological response (lowering of blood glucose levels in animal
models) equivalent to greater than 80% of that achieved by insulin
delivered subcutaneously but clinical trials have not started yet [72].
Upregulation of the expression of some receptors following the
appropriate stimulant is another strategy for nanoparticles targeting.
Stimulation of these receptors with ligands, LPS or cytokines,
increases particle uptake by the FAE cells [73].
The composition of the nanoparticles will influence their fate
within the enterocytes or M cells. While non biodegradable
nanoparticles such as polystyrene nanoparticles are not modified, it
is not clearly understood if the nanoparticles made of so called
biodegradable polymer remain intact. Both in vitro on Caco-2
monolayers and in vivo imaging studies suggest that nanoparticles
can be transcytosed through the enterocyte monolayer (Fig. 4).
Efflux pumps may strongly reduce the extent of net drug uptake
[74]. Indeed, intestinal epithelial membrane expresses ATP-binding
cassette transporters such as P-glycoprotein (P-gp), multi-drug
resistance-associated proteins in addition to various solute carrier
transporters [75]. The ATP-binding cassette (ABC) transporters family
acts in an ATP dependent manner and can pump against a steep of
concentration. So ABC transporters may reduce the amount of drug
absorbed and limit bioavailability in a dose-dependent, inhibitable
and saturable manner [76]. When drugs are encapsulated in
polymeric nanoparticles, they remain mainly associated with the
particles and are not likely to be substrate of the efflux pump. In
contrast, hydrophobic drugs loaded in the core of polymeric micelles
can be released from the micelles and are more likely to be
transported by the efflux pumps. Moreover, some polymers forming
polymeric micelles inhibit the Pgp. Pluronics, TPGS and PEG-PCL
inhibit Pgp and enhance net drug transport through intestinal barrier
[3,37]. The required structure for Pgp inhibition by a polymer and its
mechanisms of inhibition are still unclear. For instance, the inhibition
of Pgp by Pluronics, maximal just below CMC, is associated with an
increase in membrane fluidity and decrease in ATPase activity
whereas PEG-PCL inhibits Pgp above CMC [3].
The mechanisms of transport of the polymeric nanoparticles and
polymeric micelles have been schematized in Fig. 4 and summarized
in Table 3.
3.4. Pharmacokinetics and biodistribution of nanocarriers administered
orally
As discussed, size, composition, surface characteristics and architecture of the polymeric nanocarrier are determinant for the optimization of
Fig. 4. Schematic representation of the fate of polymeric nanoparticles and micelles for oral drug delivery. Different pathways for transport of nanocarriers or drug through
enterocytes or M cells are represented by orange (for nanoparticles) and blue (for micelles) arrows. (1) receptor mediated endocytosis (2) non specific transcellular transport (3)
paracellular transport (4) M cell mediated transport. Size of arrows represents contributions of each kind of transport.
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L. Plapied et al. / Current Opinion in Colloid & Interface Science 16 (2011) 228–237
Table 3
Mechanisms of nanocarrier transport across intestinal epithelium.
Mucoadhesion
Paracellular transport: tight
junction opening
Passive diffusion
Fluid phase endocytosis
Receptor mediated endocytosis
Efflux pump inhibition
Nanoparticles
Micelles
Variable (chitosan+)
Variable (chitosan+)
−
−
−
+
If specific ligand
Escape
+ for unimers and drug
+ for micelles
Variable
oral formulations: they influence nanocarrier stability and uptake by
enterocytes or M cells. After absorption, drug and/or drug loaded
nanocarrier can be included in cytoplasmic vesicles or diffuse in the
cytoplasm and be discharged in the serosal spaces to gain access to the
mesentheric lymph or blood.
Water-insoluble polymers forming stable nanocarriers (e.g. PLGA)
are more likely to be absorbed as particles whereas polymers forming
less stable particles forming polyelectrolyte complexes (e.g. chitosan)
or polymeric micelles will partly dissociate and will not be completely
absorbed as a particle. Whether the polymer itself will be absorbed
will depend on the physicochemical properties of the polymer e.g. its
molecular weight, conformation, and hydrophobicity.
When taken up by M cells, nanoparticles will be transcytosed close
to immune cells and are more likely to be delivered to the GALT and
lymphoid cells [7]. In contrast, nanoparticles, micelles or drugs taken
up by absorptive enterocytes will be mainly delivered in the blood.
The characterization of both M cells and enterocytes absorption and
crossing to the blood and lymph vessels has not been systematically
analyzed.
Once absorbed in the blood, the chemical and physical properties of
the nanoparticles which are essential parameters for oral absorption will
also affect pharmacokinetics and biodistribution. The factors that
influence their pharmacokinetics have been recently reviewed [77].
They include (i) surface modification with PEG to avoid uptake by the
reticulo-endothelial system (RES) and prolong circulation half-life,
(ii) small size to decrease uptake by RES and allow diffusion in the
tissues and (iii) neutral charge. Therefore, whether the drug is absorbed
either free or encapsulated is essential to assess.
Compared to the high amount of in vitro studies described in
literature, the oral delivery of peptides and proteins or vaccine using
polymeric nanoparticles in vivo has been less described. Moreover,
most of the studies, in particular insulin, focus on the evaluation of the
plasma pharmacokinetics of the drug and/or its therapeutic or
immune response. The fate of the nanoparticles and the polymers is
neither well understood nor investigated. Nevertheless, recently
published papers using advanced imaging and analytical technologies
give new insights on their fate. Data cannot be compared and lead to
sometimes controversial conclusions. A few selected examples
indicate that the techniques are now available for a better understanding of the fate of particles.
Oral delivery of pH-responsive nanoparticles composed of chitosan
and poly-glutamic acid loaded with aspart-insulin was studied by
single-photon emission computed tomography. Insulin was absorbed
into the systemic circulation while the carrier chitosan was mainly
retained in the oral tract [78]. PLGA nanoparticles delivered orally
were detected after 7 days in several organs including liver, spleen,
lungs, brain and kidneys. Most of the particles were located in the liver
[79]. PEGylated PLGA-based nanoparticles were rapidly taken up by
peritoneal macrophages [79]. Multilayered nanoparticles showed
co-localization in the small intestinal mucosa of insulin and alginate
[80].
Oral application of polymeric micelles is not commonly studied and
the fate of micelles is generally not investigated as the majority of
micellar systems are being developed for injections rather than oral
administration. Due to their dynamic structure, the mechanisms of drug
235
absorption after oral delivery of drug loaded polymeric micelles differ
from nanoparticle uptake. Indeed, both the micelles and the free drug
released from the micelles can be absorbed. Above the critical micellar
concentration (CMC,) the drug encapsulated in micelles and the free
drug can be absorbed whereas below CMC, the drug is released by
micelle disassembly. Hence, both micelles and unimers can be absorbed
in the systemic circulation. Oral delivery of PEG-p(CL-co-TMC) resulted
in 40% absorption of the polymer [39,40]. Pluronic-PAA copolymers
demonstrated that these molecules are excreted when administered
orally and do not absorb into the systemic circulation [1].
4. Discussion and conclusion
During the last years, polymeric nanocarriers have been studied
for oral drug delivery at preclinical level to establish proof of concept
that they can be useful to deliver drugs orally. The reasons for this
increasing interest result from the unmet medical needs that must be
addressed. Oral delivery of biopharmaceutical macromolecules
(proteins, monoclonal antibodies, and vaccines) by nanoparticles
could offer a promising alternative to parenteral administration for a
patient-friendly, needle-free delivery. Indeed, many publications and
patents demonstrate that polymeric nanoparticles enhance the
bioavailability of biopharmaceuticals in preclinical models. Whether
these biopharmaceuticals-loaded nanoparticles will be marketed in
the future remains uncertain: (i) the polymer synthesis and
nanoparticle manufacture could be too expensive for marketing and
scaling-up purposes, (ii) even if significantly improved, the bioavailability could be still too low to reach stable and efficient therapeutic
levels, (iii) the inter- and intra-individual variations in pharmacokinetics could be too high for therapeutic proteins, and (iv) the optimal
polymeric composition of the nanoparticle for a specific drug is not yet
defined: both particles made of water insoluble polymers and
polyelectrolyte complexes have shown promising preclinical results
with potential advantages and disadvantages in terms of drug
encapsulation and stability in GI tract, cells or blood. For vaccine
delivery, the PLGA-based nanoparticles targeting M cells might be
more efficient as they deliver the antigen directly to the immune cells
and might achieve sustained release [70]. For the delivery of
therapeutic peptides and proteins, the selection of a nanocarrier is
more controversial. Promising preclinical results have been reported
using chitosan-based nanoparticles [18], vitamin B12 coated nanoparticles [72]. In contrast, self assembling polymeric micelles might be
a viable approach for the delivery of poorly soluble drugs and could be
part of the decision tree in the pharmaceutical development of new
chemical entities concomitantly with cyclodextrins, solid dispersions
or lipid-based systems.
The recent findings suggest that more sophisticated nanosystems
will be developed for the oral delivery of drugs, biopharmaceuticals
and vaccines, based on the recent advances in the (i) development of
biocompatible polymers with tailored properties for oral drug
delivery and formulation of nanocarriers, (ii) the understanding of
cellular uptake mechanisms of polymeric nanocarriers (Fig. 4),
(iii) the novel techniques to study the fate of nanocarriers, polymers
and drugs in the body and (iv) the identification of new ligands for
targeted oral delivery. If the mechanism(s) of transport of the
nanocarriers through the intestinal barrier has been well characterized,
the fate of these nanocarriers after oral delivery should be investigated
with novel tools to determine how the carrier, the polymer(s) and the
drug are absorbed, biodistributed and eliminated.
These advances highlight the need to tune and to control the design
and the manufacturing of “art drug delivery systems”. Indeed, an “in
depth review” of the literature underlines the necessity of a balance in
the physicochemical properties of the nanocarriers [13]. Their size
should be small (10 to maximum 200 nm) to promote diffusion in the
mucus and uptake by intestinal cells and to decrease uptake by RES.
However, a small size is associated with a smaller drug cargo. The
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mucoadhesion should prolong the residence time without impeding
diffusion in the mucus. Surface charge should be positive to favour
interaction with mucus and cell membrane but neutral to decrease RES
clearance. The nanoparticles should remain stable in the GI tract while
releasing the drug at the appropriate site and at appropriate rate.
These novel nanocarriers might be useful to address specific needs.
Nanoparticles could be targeted to M cells or immunocompetent cells
in the GI tract for oral immunisation [7,70,81]. The oral delivery of
therapeutic peptides and proteins is the “holy grail” for formulation
scientists. If many preclinical data report a low but significantly
enhanced bioavailability of protein, the feasibility of oral nanoparticular delivery of proteins remains open and should be investigated
with optimized systems. In case of inflammatory bowel disease,
nanoparticles could also be targeted to inflammatory areas to deliver
topically either conventional small drugs or biopharmaceuticals. The
oral delivery by polymeric micelles of poorly soluble drugs, in
particular anticancer drugs which could be passively target by the
Enhanced Permeabilisation-Retention [82] effect, is a promising route.
Acknowledgment
L. Plapied and A. des Rieux are supported by Fonds National de la
Recherche Scientifique (FNRS, BE) and N. Duhem by Wallonia (BE).
We thank FRSM for financial support.
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